The present invention generally relates to the repair of damaged tissues in animals and particularly humans, and, more particularly, to the modulation of the healing of wounds in such tissue.
Injury to animal tissue resulting in tissue wounds occurs from an endless variety of pathological and non-pathological causes. In response to injury, a variety of cells have been determined to cooperate to repair the damaged tissue and heal the wound. Cells resident in the local tissue participate, as do circulating blood cells specifically recruited into the wound itself and the area nearby. Dramatic changes in cellular function are required by both the resident and recruited cells in order to initiate, coordinate, and sustain the complex process of wound healing. Damaged cells and disrupted tissue matrix must be removed, new cells must be born, and must grow and mature to replace those lost. The tissue matrix must be resynthesized and remodeled, and even the microvasculature may need to be rebuilt to supply the new tissue. It is now recognized that cytokines exchanged among responding cells mediate the induction, control, and coordination of these and other cellular functions necessary to successfully heal the wound.
Among recruited cells, macrophages are considered essential for normal wound healing. Macrophages are a rich source of peptide cytokines, which, as a group, are thought to be integral to the tissue repair responses to local injury. It is well known that individual cytokines can act on more than one cell type and can have more than one effect. New cytokines continue to be described, and new functions are being attributed to them, as well as to previously described cytokines.
Attention has recently been focused on the potential therapeutic role of a number of cytokines in the acceleration of normal wound healing, as well as in the treatment of difficult, chronically non-healing wounds. The cytokines under study include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), the transforming growth factors .alpha.0 and .beta., and cachectin/tumor necrosis factor-.alpha. (TNF). Many cytokines are delivered locally at the wound site by recruited macrophages, which function as the primary scavengers of debris and secrete a large variety of chemotactic, effector, and growth factors.
Accordingly, Fahey et al. supra conducted certain studies with a murine model of wound inflammation to determine the time course of appearance, if any, exhibited by certain cytokines, among them cachectin/TNF, interleukin-1, MIP-1.alpha., MIP-1.beta. and MIP-2. The murine model utilized by the investigators included an artificial "wound chamber" consisting of a length of perforated silicone tubing containing within its bore a length of polyvinylalcohol sponge. This wound chamber was then inserted into surgically produced subcutaneous pockets in mice. The investigators noted that inflammatory cells rapidly appeared in the recovered wound chamber fluid; and that when the wound chambers were recovered after a few days in situ, fibroblasts had colonized the recovered sponges, collagen and other tissue components had been deposited around the implant and new blood vessels had likewise formed. All of the events noted reflect the natural progression of the inflammatory phase of cutaneous wound healing.
The investigation also revealed that the levels of cachectin/TNF and IL-1 peaked on the first day after the implantation of the wound chamber, and that MIP-1 and MIP-2 were detected on day 3 of implantation only. The data thus suggested that the noted cytokines appear in the early inflammatory response in wound healing.
The novel cytokines, macrophage-inflammatory protein 1 (MIP-1) and macrophage-inflammatory protein 2 (MIP-2), have been previously identified and implicated as mediators of inflammation. MIP-1 is a heparin-binding protein of about 8000 Daltons, which is secreted in large amounts by stimulated macrophages and which migrates as a doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The two electrophoretic bands were resolved, and the corresponding peptides were partially sequenced independently, as set forth in application Ser. No. 104,287. Complete cDNAs coding for the two peptides were thereafter sequenced, and the translated peptides have been designated MIP-1.alpha. and MIP-1.beta., as set forth in application Ser. No. 238,937. Purified doublet MIP-1 has been shown to be a potent pyrogen (See also Davatelis, G. et al., "Macrophage Inflammatory Protein-1: A Prostaglandin-independent Endogenous Pyrogen", Science, 243:1066-1068, 1989) and to activate neutrophils as shown by enhancing neutrophil chemokinesis and generation of a superoxide burst (See also Wolpe, S. D. et al., "Macrophages Secrete A Novel Heparin-binding Protein With Inflammatory and Neutrophil Chemokinetic Properties", J. Exp. Med., 167:570-582, 1988).
The inflammatory cytokine macrophage-inflammatory protein 2 (MIP-2) was disclosed in co-pending application Ser. No. 240,078. This inflammatory cytokine was found to bind to heparin more avidly than MIP-1 and exhibited the distinguishing characteristics over the former of a molecular weight of approximately 6 kilodaltons, and chemotactic rather than chemokinetic activity for neutrophils. The cytokines MIP-1 (doublet), MIP-1.alpha. and MIP-1.beta. and MIP-2 were later determined to exhibit a promoting effect on the colony and cluster formation activities of granulocyte-macrophage progenitor cells (CFU-GM) from the bone marrows of normal mice and humans co-stimulated with suboptimal concentrations of known colony stimulating factors. In co-pending application Ser. No. 377,937, the utility of these cytokines in promoting myeloid blood cell production has been demonstrated.
The exact role that cytokines such as MIP-1, MIP-1.alpha., MIP-1.beta. and MIP-2 play, if any, in the promotion and facilitation of wound healing remains to be determined, and it is to this determination that the present Application is directed.